This is a proposal to identify and study the mechanism of cholinergic inhibition of cochlear hair cells using physiological, pharmacological, and molecular genetic analyses. We will also begin to examine the developmental regulation of this response. Efferent axons arising from cells in the brainstem provide an inhibitory input to outer hair cells in the mammalian and avian cochleas. This inhibitory effect is thought to result from the release of acetylcholine (ACh) from the efferent endings. Several lines of evidence point to the fact that the cholinergic response of cochlear hair cells may be mediated by a functionally unique ACh receptor (AChR). We propose to study the cholinergic response of hair cells isolated from the cochlea of the chick in an effort to understand the cellular and molecular bases of this inhibition. In addition, we intend to study the developmental acquisition of sensitivity to ACh in order to learn what factors govern the selective expression of these receptors in outer hair cells in adults. The experiments to meet these goals will be largely electrophysiological in nature, reflecting the established expertise of this laboratory. These will include whole-cell, cell-attached and excised patch voltage-clamp recordings. Also, we will use scanning confocal microscopy of intracellular signals from Ca-indicator dyes in order to assess the role of Ca as a second messenger in the hair cell response to ACh. The initial goal of our experiments in molecular biology will be to identify the genes coding for the hair cell AChR. We will first screen cochlear mRNA by RNAase protection using known brain alpha subunit clones. Another endeavor will be based on the polymerase chain reaction (PCR) to amplify cochlear cDNA that contains sequences coding for the bungarotoxin binding site in AChRs. In this way we may be able to identify a potentially unique cochlear AChR. The developmental acquisition of AChRs by hair cells will be studied during the last 10 days of embryogenesis, that is, preceding and spanning the time that efferent axons make synapses in the cochlea. Initially we will ask: when sensitivity to ACh arises, whether it is identical to mature responses, and whether all hair cells, or only presumptive outer hair cells, are sensitive? Later experiments will examine the effect of denervation and the role of activity in AChR acquisition. The motivation for this study is multi-part: to better understand the mechanism of cochlear inhibition, to contribute to the study of the molecular biology and biophysics of AChRs, and to begin to study the factors governing the developmental maturation of hair cell sensitivity to ACh.